This invention relates to human skin fibroblast collagenase and, more particularly, to the cDNA clone representing the full size human skin fibroblast collagenase mRNA.
Collagens constitute the most abundant proteins of the extracellular matrix in mammalian organisms. Though collagen turnover is generally very slow, its metabolism intensifies dramatically concomitant with processes requiring remodeling of the connective tissue, such as uterine involution, bone resorption, and wound healing. Enhanced collagen metabolism has also been implicated in the pathogenesis of a number of diseases which include recessive dystrophic epidermolysis bullosa, rheumatoid arthritis, corneal and gingival disease. The initiation of the dismantling of an existing collagen network requires specific enzymes, designated collagenases, which catalyze the initial step in the proteolytic degradation of collagen.
Several types of collagenase can be distinguished based on their physical properties and substrate specificity for different types of collagen. Collagenases degrading the interstitial collagens, types I, II and III, do not cleave collagen types IV and V, which apparently are degraded by other proteases. The structural relationship among these functionally different collagenases has not yet been determined. The interstitial collagenases from human skin synovium, gingiva and monocytes comprise a group of metalloendoproteases which generally appear to be similar. Human granulocyte collagenase, which also degrades interstitial collagens, differs from the other interstitial collagenases immunologically, in substrate preference, and in molecular weight, indicating that tissue differences in human interstitial collagenases exist. All these enzymes catalyze a single specific cleavage in each of the three collagen polypeptide chains, thereby rendering the collagen fiber soluble, thermally unstable and susceptible to attack by specific gelatinases and probably by other tissue proteases.
Collagenase from human skin fibroblasts has been purified as described by Stricklin et al., Biochemistry 16, 1607-1615 (1977), Ibid., 17, 2331-2337 (1978). The proenzyme is secreted as two closely related polypeptides reported to have apparent molecular weight of 60 kilodaltons (kDA) and 55 kDA, respectively. Both enzyme forms can be activated by several different mechanisms to produce active enzyme. See Tyree et al., Arch. Biochem. Biophys. 208, 440-443 (1981) and Stricklin et al., Biochemistry 22, 61-68 (1983). Collagenase from human skin fibroblasts also has been characterized enzymatically by Welgus et al., J. Biol. Chem. 256, 9511-9515 (1981).
Further background information on mammalian collagenase can be had by reference to a treatise such as, for example, Collagenase in Normal and Pathological Connective Tissues, Woolley and Evanson, Eds., John Wiley & Sons, New York, N.Y., 1980.
Recent advances in biochemistry and in recombinant DNA technology have made it possible to synthesize specific proteins, for example, enzymes, under controlled conditions independent of the organism from which they are normally isolated. These biochemical synthetic methods employ enzymes and subcellular components of the protein synthesizing systems of living cells, either in vitro in cell-free systems, or in vivo in microorgamisms. In either case, the principal element is provision of a deoxyribonucleic acid (DNA) of specific sequence which contains the information required to specify the desired amino acid sequence. Such a specific DNA sequence is termed a gene. The coding relationships whereby a deoxyribonucleotide sequence is used to specify the amino acid sequence of a protein is well-known and operates according to a fundamental set of principles. See, for example, Watson, Molecular Biology of the Gene, 3d ed., Benjamin-Cummings, Menlo Park, Calif., 1976.
A cloned gene may be used to specify the amino acid sequence of proteins synthesized by in vitro systems. DNA-directed protein synthesizing systems are well-established in the art. Single-stranded DNA can be induced to act as messenger RNA (mRNA) in vitro, thereby resulting in high fidelity translation of the DNA sequence.
It is now possible to isolate specific genes or portions thereof from higher organisms, such as man and animals, and to transfer the genes or fragments to microorganisms such as bacteria or yeasts. The transferred gene is replicated and propogated as the transformed microorganism replicates. Consequently, the transformed microorganism is endowed with the capacity to make the desired protein or gene which it encodes, for example, an enzyme, and then passes on this capability to its progeny. See, for example, Cohen and Boyer, U.S. Pat. Nos. 4,237,224 and 4,468,464.